Inhibition of STAT1α activity in STAT1β overexpressing cells. (A) Expression and phosphorylation status of STAT1α and STAT1β. Phosphorylation status of STAT1α and STAT1β was assessed using Western blotting with an antiphosphotyrosine 701 STAT1 antibody. Cells were treated (+) (lanes 5, 6, 9, and 10) or not treated (–) (lanes 3, 4, 7, and 8) with doxycycline for 24 hours. Parental (lanes 1 and 2) and transfected (lanes 3-6) PRI cells were treated (lanes 2, 4, 6, and 10) or not treated (lanes 1, 3, 5, 7, and 9) with IFN-γ (250 U/mL) for 30 minutes (lanes 2, 4, and 6) or for 24 hours (lanes 9 and 10). Note that lanes 3 and 7 and lanes 5 and 9 represent identical samples from 2 independent experiments. (B) Analysis of the binding activity of the STAT1 isoforms to the SIE DNA probe. The binding activity to SIE was assessed using EMSA with 10 μg nuclear protein extract from parental (lanes 1-3) and transfected (lanes 4-9) cells that were either treated (+) (lanes 5, 7, 8, and 9) or not treated (–) (lane 4) with doxycycline for 24 hours and either treated (+) (lanes 2, 3, 5, 7, 8, and 9) or left untreated (–) (lanes 1 and 4) with IFN-γ (250 U/mL). Nuclear extracts were obtained from the cells shown in panel A, lanes 1 to 6. Extracts were incubated with the radiolabeled m67-SIE probe and separated on a polyacrylamide gel, and radioactivity was determined using a phosphor imaging screen. Supershifts were performed using antibodies recognizing either STAT1α and STAT1β (lanes 3 and 9) or STAT1α alone (lane 8). Supershifts show that the lower band (lane 7) comprises only STAT1β homodimers, whereas the upper bands (lanes 1, 2, and 5) comprise STAT1α homodimers and STAT1α/ STAT1β heterodimers, respectively. Note that a/a, a/b, and b/b represent the 3 different complexes binding specifically to the SIE probe. NS indicates nonspecific binding; and BS, supershifted bands (lanes 3, 8, and 9).